Cardiac progenitor cells (CPCs)?identified in early embryos?are cell sources to make the heart during fetal development, and abnormal CPC development is closely associated with the etiology of congenital heart defects. In particular, proper regulation of their number and fate is essential for the ensuing heart growth. However, it remained unknown if they undergo self-renewal and if there is a dedicated environment for their maintenance. We have identified a renewing population of CPCs and their niche during development, and this proposal aims to elucidate the mechanisms governing the self-renewal and expansion of CPCs in vivo. Numb family proteins (NFPs) are conserved endocytic proteins with critical roles in cell-fate decisions. We found that precardiac deletion of NFPs depletes CPCs in the second pharyngeal arch (PA2) and is associated with a hypoplastic heart and early embryonic lethality. Based on this phenotype, we hypothesized that CPCs expand in the PA2 before their cardiac differentiation. Confirming the hypothesis, CPCs normally remained undifferentiated and expansive in the PA2, and differentiated into cardiac cells soon as they migrated out of the PA2. CPCs co-cultured with PA2 cells formed distinct colonies that continued to grow without cardiac differentiation, and differentiated into cardiac cells when PA2 cells were removed. These suggested that CPCs proliferate without differentiation in the PA2, suggesting the presence of a stem cell?niche paradigm. The proliferation was promoted by Hedgehog (Hh) proteins, crucial developmental and stem cell regulators enriched in the PA2, implying the Hh signaling may mediate the environmental role of PA2 cells. To determine the CPC-autonomous role of NFPs in the PA2, we generated lineage-specific mosaicism that allowed tracing of CPCs lacking NFPs without causing the lethality. The NFP-deleted mutant CPCs normally populated in the PA2, but failed to expand in the PA2 and progressed to cardiac cells. The mutant CPCs showed dramatically decreased levels of the conserved stem cell regulator Itgb1. Itgb1 physically associated with NFP in CPCs, and precardiac deletion of Itgb1 resulted in CPC depletion in the PA2, similar to the phenotype of NFP-deleted embryos. These findings set the stage for a mechanistic exploration of CPC maintenance by intrinsic and extrinsic factors. The specific aims of this proposal are (1) to determine if NFPs play an instructive role for CPC renewal in the PA2, (2) to determine if NFPs prevent endolysosomal degradation of Itgb1 for CPC maintenance, and (3) To investigate if PA2 cells affect CPC renewal and expansion via Hh signaling. With these aims, I expect to elucidate the factors and mechanisms by which CPCs are maintained in a renewing state in their microenvironment. This knowledge will provide first insights into the mechanistic understanding of the self-renewal of CPCs in their microenvironment during heart development, which will open up new avenues of research in congenital heart disease and may allow us to maintain and expand CPCs in a homogeneous and undifferentiated state in vitro for heart regeneration research.